Process for the preparation of riboflavin

ABSTRACT

A process is provided for preparing riboflavin from D-glucose in good yield and under efficient conditions.

BACKGROUND OF THE INVENTION

The present invention relates to a new process for the preparation ofriboflavin starting from D-glucose.

Processes for the preparation of riboflavin are known. The processusually is carried out on a large industrial scale and uses D-glucose asthe starting material, which is oxidized to D-arabonic acid, theD-arabonic acid is epimerized to D-ribonic acid and the D-ribonic acidis then converted into D-ribonolactone, from which D-ribose is formed byamalgam reduction, the D-ribose being hydrogenated toN-D-ribityl-3,4-xylidine via xylidine riboside. Riboflavin can then beobtained from the N-D-ribityl-3,4-xylidine by reaction with diazotizedaniline and barbituric acid.

The yields for the stages from D-glucose to N-D-ribityl-3,4-xylidine inthis process are about 20-23%. This results in a total yield ofriboflavin, starting from D-glucose, of 15-16% of theory. Besides therelatively poor total yield, the disadvantage of this process is alsothe epimerization stage from D-arabonic acid to D-ribonic acid, in whicha number of resinous by-products are formed which make a complicatedpurification necessary. However, the amalgam reduction in particularpresents enormous problems, since when large amounts of mercury areutilized, a considerable effort must be made to keep both the productsand the waste substances free from mercury.

Processes which avoid the use of mercury have therefore already beenproposed. Thus, D-ribonic acid or D-ribonolactone can be hydrogenated toN-D-ribityl-3,4-xylidine in the presence of 3,4-xylidine or4-nitro-1,2-xylene in one stage. However, this process requires a verycapital-intensive high pressure hydrogenation unit, since thehydrogenation proceeds under a pressure of about 250-300 bars. In spiteof saving intermediate stages and in spite of the high expenditure, theyield of N-D-ribitylxylidine in this process, starting from D-glucose,is also only about at most 35%, which results in a riboflavin yield ofless than 25%.

It has furthermore been proposed to prepare D-ribose directly fromD-glucose by fermentation in a microbiological process with the aid ofsuitable microorganisms and then to obtain N-D-ribityl-xylidine andriboflavin from the D-ribose in the customary manner. Although thisroute has advantages, considerable difficulties which impair theprofitability of the process are nevertheless to be expected when thebiochemical process, which is complicated and above all susceptible todisturbances, is carried out on a large industrial scale. Moreover, theyield of N-D-ribityl-xylidine in this process, starting from D-glucose,is also only about 34%, which results in a riboflavin yield of about24%.

OBJECTS OF THE INVENTION

It is, therefore, an object of this invention to provide a process forthe preparation of riboflavin which leads to a good yield of a pureproduct in simple reaction steps which can be carried out without greatinvestment, using inexpensive starting materials and avoiding highlytoxic reactants which pollute the environment.

Upon further study of the specification and appended claims, furtherobjects and advantages of this invention will become apparent to thoseskilled in the art.

SUMMARY OF THE INVENTION

These objects have been attained by providing a process for thepreparation of riboflavin from D-glucose, comprising the steps of:

(a) oxidizing D-glucose to D-gluconic acid or an alkali metal gluconate;

(b) oxidizing the resultant D-gluconic acid or gluconate to D-arabinosewith hypochlorite;

(c) epimerizing the resultant D-arabinose to D-ribose, catalyzed by amolybdenum (VI) compound;

(d) catalytically hydrogenating the resultant D-ribose in the presenceof 4-nitro-1,2-xylene or 3,4-xylidine to form N-D-ribityl-3,4-xylidine;

(e) coupling the resultant N-D-ribityl-3,4-xylidine with abenzenediazonium salt to form1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene; and

(f) reacting the resultant1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene with barbituric acid toform riboflavin.

DETAILED DISCUSSION

The process of the invention uses D-glucose as the starting material.However, it does not proceed via arabonic acid and ribonic acid, butinstead gives D-ribose and N-D-ribityl-3,4-xylidine via gluconic acidand arabinose. It avoids the disadvantages of the known processes andleads to even higher yields of riboflavin.

The individual stages can be carried out without difficulty, usinginexpensive and non-toxic reactants.

In some cases, isolation of intermediate products can be dispensed with,without losses in yield, so that the end product is obtained withoutexcessive expenditures of work and capital.

While the individual steps of the process according to the invention arebroadly familiar to the art, it could not be predicted that preciselythis combination of individual reactions would lead to such anadvantageous overall result, particularly since numerous attempts havealready been made to prepare this important product in an advantageousmanner on a relatively large scale. In particular, it could not beenvisaged that the overall concept, which was already very advantageous,could be improved further by inventive improvements to numerousindividual steps of this combination.

It is particularly surprising that substantially higher yields thanthose obtained by the processes known hitherto are neverthelessachieved. Thus, in the process according to the invention,N-D-ribityl-xylidine is obtained, starting from D-glucose, in a yield ofabout 40%, which corresponds to a riboflavin yield of almost 30%,relative to D-glucose.

As in the case of the known processes, readily accessible, inexpensiveD-glucose can be used as the starting material for carrying out theprocess. D-gluconic acid can be obtained therefrom in a known manner, byfermentative, chemical or electrochemical oxidation, in very high yieldsof over 90%. A summary of such processes can be found, for example, inInd. Eng. Chem. 11, 370, (1972).

Fermentative oxidation is preferably carried out. For this, D-glucose istreated with a suitable strain of bacteria, such as, e.g., Acetobactersuboxydans, under sterile conditions in aqueous solution, to whichnutrients are added. The reaction is effected in a fermenter, whileaerating the mixture and neutralizing the acid formed with a base,preferably sodium hydroxide solution, until the test for sugar gives anegative result, i.e., all the glucose has reacted. The oxidation isgenerally complete after about 10 to 40 hours, depending on the size ofthe batch and the bacterial strain.

The fermentation reaction effects a substantially quantitative oxidationof the D-glucose. After filtration or centrifugation to remove cellularmatter, the resultant solution, generally containing about 20-35% byweight of gluconate, is advantageously used directly for the subsequenthypochlorite oxidation to arabinose, although solutions of higherconcentration can also be used.

The known oxidation of gluconate to arabinose by sodium hypochlorite,reported in J. Am. Chem. Soc., 81, 5190 (1959), was carried out in verydilute solution and at relatively low temperatures, because of thepresumed instability of the hypochlorite. The oxidation, for which about2.5 times the theoretical amount of hypochlorite was employed, proceededover a period of 20-30 hours and gave a yield of only about 40%.

It has now been found that the oxidation of gluconate, preferably analkali metal gluconate, to arabinose with hypochlorite can besubstantially improved if it is effected at a high gluconateconcentration and a high temperature. Surprisingly, the yield ofarabinose resulting from the present process is about double that of theprior art process, by using these unexpectedly superior conditions.Moreover, since the reaction time of about 20-30 hours can also beshortened to about 10-60 minutes in the process according to theinvention, a space/time yield which is improved by a power of ten isachieved in the present process.

It is particularly surprising that arabinose is produced in such a highyield under the present conditions, since arabinose may itself befurther oxidized and/or degraded by hypochlorite under these sameconditions. Despite the known instability of hypochlorite, whichdecomposes very readily with evolution of chlorine, especially in acidsolution, or is disproportionated to chlorate and chloride in neutralsolution, the oxidation reaction can evidently compete quite well withthese decomposition reactions.

To effect the hypochlorite oxidation, a gluconate solution is used,preferably an alkali metal gluconate, and preferably the resultantsolution from fermentative oxidation of D-glucose, the gluconate beingat a concentration of from 10 to 40% by weight in aqueous solution,preferably from 20 to 35% by weight. The gluconate solution is broughtto a temperature of between about 30° and about 90° C., preferably toabout 50°-70° C., and is adjusted to a pH value of about 4-6, preferablyabout 4.5-5.5, with an acid, preferably a mineral acid, and mostpreferably with hydrochloric acid.

To oxidize the gluconic acid, a relatively concentrated aqueoushypochlorite solution is then run into the gluconate solution, the pHvalue of the reaction solution being kept substantially constant bysimultaneously adding acid, preferably hydrochloric acid. From 1.0 to1.5 equivalents of hypochlorite are used, preferably 1.1-1.2equivalents. The concentration of the hypochlorite solution is from 10to 20% by weight. Despite the fact that the present process usesconditions which permit a rapid conversion and throughput on a largeindustrial scale, the oxidizing agent is nevertheless very much betterutilized than in the prior art, where a higher ratio of hypochlorite togluconate was required.

Any available hypochlorite may be used, but preferably sodium, potassiumor calcium hypochlorite is employed. Advantageously, the hypochloritesolution is prepared by passing chlorine into a solution of sodium,potassium or calcium hydroxide.

The hypochlorite solution is metered into the warm gluconate solution ata rate such that the addition is complete within a period of about 10-60minutes, preferably about 30 minutes. The hypochlorite solution can, ofcourse, also be metered in over a longer period, and this may beunavoidable in the case of very large batches. However, as a rule, theaddition is effected in as short a time as possible. Thereafter, themixture is stirred for a further brief period until the excesshypochlorite has disappeared. D-arabinose may then be isolated in aconventional manner. The yield of D-arabinose is about 70-75%, relativeto the starting D-glucose, and this is achieved even when the crudefermentation solution from oxidation of D-glucose is employed.

In order to isolate the D-arabinose, it is known to concentrate thereaction solution, whereupon most of the sodium chloride precipitatesand can be separated off, and then to free the solution from theresidual amount of ionic constituents by ion exchange.

A particularly advantageous method, which is also an aspect of thepresent invention, is to separate off ionic constituents from thereaction solution with the aid of electrodialysis. This process offerssubstantial technological advantages. Thus, for example, considerableamounts of chemicals which would otherwise be required to regenerate theion exchanger are saved. A surprising advantage is also that, during theelectrodialysis, the ionic constituents migrate through the exchangermembrane together with a hydrate shell and a considerable amount ofwater is thus simultaneously removed from the reaction solution. Sincethe reaction solution must be concentrated anyway to obtain thearabinose, considerable amounts of energy are thereby saved.

Conventional electrodialysis cells may be used for this process, and thearabinose-containing solution is circulated through the cell whilecurrent is applied, typically at a voltage of, e.g., from 1 to 3 voltsper subunit consisting of anion- and cation-exchanger membrane and acurrent strength of from 50 to 200 amps per m² for a sufficient time tofree the solution of chloride ions and other ionic by-products.Generally, all conventional measures as, e.g. described in A. T. Kuhn,Industrial Electrochemical Processes, Elsevier 1971, can be usedaccordingly.

The resultant concentrated solution is then extracted, e.g., by stirringwith methanol, whereupon a high yield of very pure D-arabinose isobtained. The overall yield, which is already very high, can beincreased further if the recovered gluconate is recycled to thehypochlorite oxidation step.

This new and advantageous route to D-arabinose from D-glucose is itselfan independently inventive aspect of the overall process of theinvention.

The D-arabinose thus obtained can be converted into an epimer mixturewhich contains D-arabinose and D-ribose in a ratio of about 3:1, otherpentoses such as D-lyxose and D-xylose, as well as other by-products, bycatalysis with molybdic acid or another molybdenum-VI compound, in aknown process. It is also known that the components of the epimermixture may be separated by chromatographic methods. However, suchseparation processes are not very suitable for the large industrialscale preparation of D-ribose because of the considerable expenditure oflabor and capital required.

Although the D-ribose, which alone is desired for further reaction togive riboflavin, only makes up a minor proportion of the epimer mixture,nevertheless, this route to ribose has proved very advantageous in thepresent process. On the one hand, the D-arabinose present as the mainconstituent of the epimer mixture can be almost completely separated offfrom the epimer mixture in a very simple manner. On the other hand, ithas been found that the epimer mixture which is obtained whenD-arabinose is epimerized under catalysis by molybdic acid or othermolybdenum (VI) compounds and which, after coarsely separating offD-arabinose, consists of the pentoses D-ribose, D-arabinose, D-xyloseand D-lyxose and other by-products, can advantageously be employeddirectly in the catalytic hydrogenation step in the presence ofnitroxylene or xylidine. Pure N-D-ribityl-3,4-xylidine can be obtainedfrom the resulting reaction mixture by crystallization. This issurprising since, because of the very great similarity of the structuresof the pentityl-xylidines formed, it was to be expected that a mixtureof ribityl-, arabityl-, xylityl- and lyxityl-xylidines would crystallizeout, it being possible to obtain the ribityl-xylidine, which alone isdesired, in a pure form from this mixture only by expensive purificationsteps. This process also represents a particularly advantageousinventive aspect of the present process.

The isomerization step is carried out by dissolving the arabinose inwater, and adding the catalyst at an elevated temperature. Theconcentration of the arabinose is not critical, but concentrations whichare as high as possible, for example, approximately 10-20% by weightsolutions, are used for good utilization of the existing equipment. Itis also possible to use directly the solution obtained in the gluconateoxidation without isolating the arabinose.

The molybdenum (VI) compound used as a catalyst is any molybdenum (VI)compound capable of epimerizing D-arabinose, preferably one which is atleast slightly soluble in water, e.g., molybdic acid and/or an alkalimetal or ammonium salt thereof. Commercially available molybdic acid isadvantageously employed.

The D-arabinose solution is heated to a temperature of about 80°-100° C.and from 0.1 to 5%, preferably 1% by weight of catalyst, e.g.,commercial molybdic acid, relative to arabinose, is added, it beingadvantageous for the pH value to be adjusted to 1-4, preferably about 3.The epimer equilibrium is established more rapidly the higher the amountof catalyst and the higher the temperature.

At a temperature of about 90°-95° C. and at a catalyst concentration of1% by weight of molybic acid, the equilibrium state is reached afterabout 2 hours. At equilibrium, a mixture of the four pentoses arabinose,ribose, lyxose and xylose is present, in addition to a certainproportion of decomposition products and oxidation products. Theequipment can be flushed with an inert gas, e.g., nitrogen, beforeand/or during the reaction in order to avoid generating an excessiveproportion of oxidation products.

When the reaction is ended and equilibrium is attained, the catalyst isremoved from the solution, e.g., with the aid of ion exchangers. Thesolution is then concentrated, preferably under reduced pressure. Mostof the arabinose present in the reaction mixture, i.e., at least 80%,desirably at least 90%, and preferably at least 95%, can be crystallizedout by adding a lower alcohol, e.g., methanol or, preferably, ethanol,and can be separated off in a pure form and recycled. This is a furtheradvantageous aspect of the present invention, since the overall yieldsare thereby increased.

The mother liquor from the crystallization can be evaporated, theresultant 10-20% of solids contained therein and recoverable therefromconsisting of D-ribose to the extent of about 75%, D-arabinose to theextent of 10%, D-xylose and D-lyxose to the extent of about 5% andby-products to the extent of about 10%. Further separation of D-ribosemay be effected by chromatography over a cation exchanger charged withcalcium ions or barium ions.

However, in the process according to the invention, this expensivepreparation of D-ribose in pure form can be omitted. Instead, the motherliquor can be employed directly in the subsequent catalytichydrogenation step.

According to the present process, the mother liquor resulting fromcrystallization of arabinose from the catalyst-free epimer mixture isdesirably diluted with water, optionally containing additional loweralcohol, to about double its initial volume. An amount in moles of4-nitro-1,2-xylene or 3,4-xylidene equal to about the total number ofmoles of pentoses in the solution is added, and catalytic hydrogenationis effected.

The hydrogenation can be effected with Raney nickel as the catalystunder a hydrogen pressure of about 50-100 bars, as described in JapanesePatent Application No. 6,665 (1964). At a hydrogenation temperature ofabout 60°-80° C., the reaction is completed after about 30-60 minutesand, after removing the catalyst and concentrating the solution, pureN-D-ribityl-3,4-xylidine crystallizes out on cooling the concentrate.

While hydrogenations catalyzed by nickel were used almost exclusively inthe processes known hitherto, it has now been found that thishydrogenation can also be carried out in a very advantageous manner withfrom 3-15% by weight, relative to the total amount of pentoses in thesolution, preferably 7-10% by weight, of 5 to 10% palladium-on-charcoal,at a low hydrogen pressure of from 2 to 5 bars, preferably about 3 bars.The hydrogenation is effected in a mixture of water and a lower alcohol,at a temperature of about 35°-80° C., the uptake of hydrogen normallyending after about 2-3 hours.

The yield is comparable to that of the high pressure hydrogenation.However, the advantages are, on the one hand, that the investment forthe hydrogenation unit is considerably lower and, on the other hand,that the catalyst can be recovered quantitatively and employed again.After-purification of the effluents to remove nickel ions can thus bedispensed with. After separating off the catalyst by filtration, thepure N-D-ribityl-3,4-xylidine is obtained in a high yield and purity oncooling the solution.

N-D-Ribityl-3,4-xylidine is the key intermediate in the synthesis ofriboflavin, through which many other known synthetic routes also pass.The conversion of this intermediate product into riboflavin is effectedin a known fashion by coupling the ribitylxylidine with abenzenediazonium salt, and then reacting the resultant azo compound withbarbituric acid. Any convenient benzenediazonium salt may be used. Aliterature survey of these known processes can be found, e.g., inKirk-Othmer, Encyclopedia of Chemical Technology, 17, 451, (1968).

Generally, phenyldiazonium chloride is added to an acidic aqueous oraqueous/alcoholic solution or suspension of the N-D-ribitylxylidine atlow temperature, and the resultant azo compound is isolated. Subsequentreaction with barbituric acid and acetic acid in solution splits offaniline to give riboflavin, which can optionally be further purified bydissolving it in aqueous hydrochloric acid, treating the solution withhydrogen peroxide and precipitating the product with water. Thisprocedure is used advantageously in the present process to convert theN-D-ribityl-3,4-xylidine obtained from the catalytic hydrogenation stepto riboflavin.

The overall yield in the process according to the invention, startingfrom D-glucose, is significantly higher than the yields achieved in theknown processes. Accordingly, the present invention provides a veryvaluable new process for the preparation of riboflavin.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following preferred specific embodiments are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. In the followingexamples, all temperatures are set forth uncorrected in degrees Celsius;unless otherwise indicated, all parts and percentages are by weight.

EXAMPLE 1

(a) Conversion of D-glucose into sodium D-gluconate.

10 l of a sterile aqueous solution which contains 1.5 kg of D-glucose.H₂O, 10 g of dry corn steep nutrient, 10 g of ammonium dihydrogenphosphate, 10 g of potassium dihydrogen phosphate and 5 g of magnesiumsulphate, and which has a pH value of 6.5, is inoculated, in afermenter, with a shaken culture of Acetobacter suboxydans ATCC 621 at atemperature of 30° C., and the mixture is aerated, while stirring. A pHvalue of 5.5-6.0 is maintained by metering in sodium hydroxide solution.After about 40 hours, when all of the glucose has been converted togluconate, the mixture is cooled and centrifuged. Sodium gluconate canbe obtained in crystalline form in a yield of over 90% by evaporatingthe centrifuged solution.

(b) Conversion of sodium D-gluconate into D-arabinose.

A solution of 218 g of sodium D-gluconate in 872 ml of water, or anamount of the fermentation solution from Example 1a containing the sameamount of gluconate, is heated to 60° C. Then, 532 ml of a sodiumhypochlorite solution with a content of 16% (weight/volume) of activechlorine are added at this temperature, the pH value being kept constantat 4.5-5.0 by simultaneously adding about 80 ml of concentratedhydrochloric acid. The solution is then freed from elecrolytes byelectrodialysis. For this, the solution is pumped, in circulation,through an electrodialysis cell with an effective membrane area of 500cm². Electrolysis is carried out at a voltage of 5 volts and at acurrent strength of 5 amperes for about 24 hours, until the arabinosesolution is completely free from chloride and free from ionicby-products. 110 g (73%) of crystalline D-arabinose can be obtained byevaporating the solution and extracting the residue by stirring withmethanol.

(c) Conversion of D-arabinose into D-ribose.

A solution of 100 g of D-arabinose in 500 ml of water, or an amount ofthe ion-free arabinose solution from Example 1b corresponding to thesame amount of arabinose, is warmed to 92° C., while flushing theapparatus with nitrogen. Then, 1 g of molybdic acid (commerciallyavailable quality, consisting mostly of ammonium molybdate) is added,and the solution is stirred for 1-2 hours. Thereafter, the catalyst isremoved by electrodialysis or by ion exchange (strongly acid and weaklybasic exchangers). The solution is concentrated to a syrup which stillcontains about 10% of water and the syrup is extracted by stirring with200 ml of ethanol. 70 g of D-arabinose crystallizes out and is separatedoff and recycled.

The aqueous/alcoholic mother liquor, which contains about 21 g ofD-ribose, 3 g of D-arabinose, 3 g of a mixture of D-lyxose and D-xyloseand 3 g of further by-products, can be chromatographed over a cationexchanger charged with calcium ions or barium ions in order to obtainD-ribose in a pure form.

(d) Conversion of D-ribose into N-D-ribityl-3,4-xylidine.

The alcoholic mother liquor from Example 1c is diluted with an equalvolume of water, the pH is adjusted to 5.8 with 5 g of sodium acetateand sufficient acetic acid. Then, 30 g of 4-nitro-1,2-xylene and 15 g ofmoist Raney nickel are added, the mixture is warmed to 80° C. andhydrogenation is carried out under a hydrogen pressure of 50 bars for0.5 hours. After filtering off the catalyst and evaporating off some ofthe alcohol, 30 g of N-D-ribityl-3,4-xylidine crystallize out oncooling.

Further N-D-ribityl-3,4-xylidine can be obtained by evaporating themother liquor, extracting the residue by stirring with 10% hydrochloricacid, whereupon only the readily soluble hydrochloride of theribitylxylidine dissolves, and neutralizing the solution which has beenseparated off from the residue, whereupon N-D-ribityl-3,4-xylidinecrystallizes out.

(e) Conversion of N-D-ribityl-3,4-xylidine into1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene.

A solution of 456 g of sodium nitrite in 1,140 ml of water is added to asuspension, cooled to -5° C., of 616 g of aniline, 1,320 ml of water and1,732 ml of 37% hydrochloric acid over the course of one hour. Thediazonium salt solution thus obtained is then allowed to run into asuspension containing 1,532 g of N-D-ribityl-3,4-xylidine, 2,200 ml ofwater, 1,800 ml of 100% acetic acid and 470 ml of 37% hydrochloric acidat a temperature of at most 5° C. over the course of 1 hour, the pHvalue being kept constant at 1.5 by simultaneously adding 32% sodiumhydroxide solution as needed. After subsequently stirring the mixturefor some time, the pH is adjusted to 3.5 with sodium hydroxide solutionand the crystals which have precipitated are stirred in the mixture atroom temperature for a further few hours and filtered off. 2,564 g ofcrude product is obtained which can be purified by recrystallizationfrom ethanol.

(f) Conversion of 1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene intoriboflavin.

A solution of 35.9 g of crude1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene, obtained according toExample 1e, and 21.6 g of barbituric acid in 135 ml of dioxane and 25 mlof glacial acetic acid is boiled for 16 hours. After cooling, theriboflavin which has precipitated is filtered off, washed with 100 ml ofwater at 50° C. and dried, 32.7 g of crude riboflavin being obtained.

The product can be purified by a procedure in which 100 g of cruderiboflavin are dissolved in 130 ml of 37% hydrochloric acid, 29 ml ofwater and 7.1 ml of 35% hydrogen peroxide at 50° C. and the filteredsolution is heated with 1,144 ml of water to 90°-100° C. for one hour.After cooling, the product is filtered off, washed with 380 ml of waterand 160 ml of methanol and dried, 88.1 g of pure riboflavin beingobtained.

The overall yield of steps a-f, relative to D-glucose, is 28% of theory.

EXAMPLE 2

The procedure followed is analogous to Example 1, but the hydrogenationis carried out as follows, instead of as described in Example 1d:

A solution of 5.243 kg of the epimer mixture from Example 1c, whichcontains about 60% of ribose, and 5.442 kg of 4-nitro-1,2-xylene in 72 lof 50% aqueous methanol (v/v) is hydrogenated at a temperature of about62° C. and under a hydrogen pressure of 3 bars using 0.6 kg of 5%palladium-on-charcoal catalyst. After about 2.5 hours, when the uptakeof hydrogen is complete, the mixture is filtered to separate thecatalyst and the filtrate is cooled to 0° C. for crystallization. Aftercentrifuging, washing and drying, a total of 4.2 kg ofN-D-ribityl-3,4-xylidine is obtained.

The recovered catalyst can be used again, after washing with glacialacetic acid and water.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

What is claimed is:
 1. A process for preparing riboflavin fromD-glucose, comprising the steps of:(a) oxidizing D-glucose to D-gluconicacid or an alkali metal gluconate; (b) oxidizing the resultantD-gluconic acid or gluconate to D-arabinose with hypochlorite; (c)epimerizing the resultant D-arabinose to obtain substantially anequilibrium epimer mixture containing D-ribose, and coarsely separatingat least 80% of the D-arabinose therefrom; (d) directly catalyticallyhydrogenating the resultant D-ribose-rich epimer mixture in the presenceof 4-nitro-1,2-xylene or 3,4-xylidine, and directly crystallizingsubstantially pure N-D-ribityl-3,4-xylidine from the resultanthydrogenation mixture; (e) coupling the resultantN-D-ribityl-3,4-xylidene with a benzenediazonium salt to form1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene; and (f) reacting theresultant 1-D-ribitylamino-3,4-dimethyl-6-phenylazobenzene withbarbituric acid to form riboflavin.
 2. The process of claim 1, whereinin step (b), an aqueous solution of D-gluconic acid is oxidized at a pHof 4-6, at a temperature of 30°-90° C. and at a D-gluconic acidconcentration of from 10-40% by weight, by rapidly adding anapproximately 10 to 20% by weight hypochlorite solution.
 3. The processof claim 2, wherein the pH is 4.5-5.5, the temperature is 50°-70° C.,and the D-gluconic acid concentration is from 20 to 35% by weight. 4.The process of claim 2, wherein the hypochlorite is sodium hypochlorite.5. The process of claim 2, wherein the pH is kept substantially constantduring the hypochlorite addition.
 6. The process of claim 2, whereinfrom 1.0 to 1.5 equivalents of hypochlorite are employed, relative tothe D-gluconic acid.
 7. The process of claim 6, wherein from 1.1 to 1.2equivalents of hypochlorite are employed.
 8. The process of claim 2,wherein step (b) further comprises electrodialyzing theD-arabinose-containing oxidation product mixture to substantially removeionic constituents, and then isolating D-arabinose from the resultantsubstantially ion-free solution.
 9. The process of claim 1, wherein instep (a), D-glucose is fermentatively oxidized using Acetobactersuboxydans, the resultant D-gluconic acid is neutralized with base, andthe resultant gluconate solution is freed of cellular matter, theinitial concentration of D-glucose being such that the resultantcell-free gluconate solution has a concentration of 20-35% by weight ofgluconate, said cell-free gluconate solution being used directly for thehypochlorite oxidation in step (b).
 10. The process of claim 1, whereinin step (c), the D-arabinose is epimerized with a molybdenum (VI)compound, and wherein the D-arabinose separated from the epimer mixtureis recovered.
 11. The process of claim 10, wherein the recoveredD-arabinose is recycled to step (c).
 12. The process of claim 11,wherein in step (c), a 10-20% by weight D-arabinose solution is heatedto 80°-100° C., 0.1-5% by weight of commercial molybdic acid relative toD-arabinose is added, the pH being adjusted to 1-4, to effect theepimerization; the catalyst is then removed, the solution isconcentrated, and a lower alcohol is added, whereby at least 90% of theD-arabinose in the epimer mixture crystallizes out and is recovered andrecycled, the aqueous alcoholic mother liquor containing D-ribose as itsmajor solute component being employed directly in step (d).
 13. Theprocess of claim 11, wherein in step (d), the D-ribose-containing epimermixture remaining after separation of at least 80% of the D-arabinosetherefrom is catalytically hydrogenated in the presence of a molaramount of 4-nitro-1,2-xylene or 3,4-xylidine equal to about the totalnumber of moles of pentoses in said epimer mixture.
 14. The process ofclaim 13, wherein the hydrogenation is effected at a temperature of35°-80° C. under a hydrogen pressure of from 2 to 5 bars, the catalystbeing palladium-on-charcoal.
 15. The process of claim 14, wherein thehydrogen pressure is about 3 bars.
 16. A process for oxidizingD-gluconic acid to D-arabinose, comprising the step of rapidly adding toa 10-40% by weight aqueous solution of D-gluconic acid at pH 4-6, at atemperature of 30°-90° C., an approximately 10 to 20% by weighthypochlorite solution.
 17. The process of claim 16, wherein the pH is4.5-5.5, the temperature is 50°-70° C., and the D-gluconic acidconcentration is from 20 to 35% by weight.
 18. The process of claim 16,wherein the hypochlorite is sodium hypochlorite.
 19. The process ofclaim 16, wherein the pH is kept substantially constant during thehypochlorite addition.
 20. The process of claim 16, wherein from 1.0 to1.5 equivalents of hypochlorite are employed, relative to the D-gluconicacid.
 21. The process of claim 20, wherein from 1.1 to 1.2 equivalentsof hypochlorite are employed.
 22. The process of claim 16, which furthercomprises electrodialyzing the D-arabinose-containing oxidation productmixture to substantially remove ionic constituents, and then isolatingD-arabinose from the resultant substantially ion-free solution.
 23. Theprocess of claim 16, wherein the D-gluconic acid is produced byfermentatively oxidizing the D-glucose using Acetobacter suboxydans, theresultant D-gluconic acid is neutralized with base, and the resultantgluconate solution is freed of cellular matter, the initialconcentration of D-glucose being such that the resultant cell-freegluconate solution has a concentration of 20-35% by weight of gluconate,said cell-free gluconate solution being adjusted to pH 4-6 and used forthe hypochlorite oxidation.
 24. A process for the preparation ofN-D-ribityl-3,4-xylidine, wherein D-arabinose is epimerized with amolybdenum (VI) compound to give an epimer mixture containing D-riboseand D-arabinose, at least 80% of the D-arabinose is then separated fromthe epimer mixture and recovered, the remaining epimer mixture isdirectly catalytically hydrogenated in the presence of4-nitro-1,2-xylene or 3,4-xylidine, and the resultantN-D-ribityl-3,4-xylidine is directly recovered by crystallization. 25.The process of claim 24, wherein the recovered D-arabinose is recycledto the epimerization step.
 26. The process of claim 25, wherein a 10-20%by weight D-arabinose solution is heated to 80°-100° C. in the presenceof 0.1-5% by weight of a molybdenum (VI) compound relative toD-arabinose, the pH being adjusted to 1-4, to effect the epimerizationto D-ribose, the catalyst is then removed, the solution is concentrated,and a lower alcohol is added, whereby at least 80% of the D-arabinose inthe epimer mixture crystallizes out and is recovered and recycled, theaqueous alcoholic mother liquor containing D-ribose as its major solutecomponent is catalytically hydrogenated in the presence of a molaramount of 4-nitro-1,2-xylene or 3,4-xylidine equal to about the totalnumber of moles of pentoses in said mother liquor, and the resultantN-D-ribityl-3,4-xylidine is recovered by crystallization.
 27. Theprocess of claim 26, wherein the hydrogenation is effected at atemperature of 35°-80° C. under a hydrogen pressure of from 2 to 5 bars,the catalyst being palladium-on-charcoal.